Vaccination of macaques with attenuated SIV strains has consistently provided the most effective protection against pathogenic SIV challenge and offers the best available experimental model to define specific mechanisms responsible for protection. Recent studies from our group have demonstrated that a significant maturation of protective immunity against vaginal challenge occurs between 5 and 20 weeks after vaccination with SIVAnef, which is associated with evolution of both cellular and humoral immune responses. Furthermore, SIVAnef-vaccinated animals do not manifest the recently described mobilization of pDCs and recruitment of CD4+ T cells that occurs early after vaginal challenge of naive animals. Based on these observations, we hypothesize that SIVAnef is able to block critical stages of SIV replication and spread in the first 7 days after vaginal challenge. Since our previous studies have largely focused on later time points (>7 days after challenge) and examination of immune responses in the peripheral blood, these emerging data dictate a new and comprehensive analysis of the early events in tissues following vaginal challenge of SIVAnef-vaccinated animals. The goal of this proposal is to apply a panel of innovative techniques to serial necropsies of SIVAnef-vaccinated animals before and after challenge to elucidate the roles of adaptive and innate immune responses in mediating protection induced by SIVAnef and to identify the sites of containment of viral replication within the female reproductive tract. Specific aims include: 1: To examine the evolution of adaptive and innate immune responses in the female reproductive tract induced by SIVAnef; 2. To identify sites of viral containment and characterize local immune responses following vaginal challenge of SIVAnef-vaccinated animals; and 3. To examine the effect of B cell depletion on protective immunity against vaginal challenge induced by SIVAnef. These studies will provide novel insights into mechanisms of protective immunity induced by SIVAnef, offer a direct comparison of both immunologic and virologic analyses for animals vaccinated with alternative vectors, and ultimately guide the identification of mechanisms of protection against mucosal infection with lentiviruses.